Related Experiment Video

Updated: Sep 28, 2026

Assaying Protein Kinase Activity with Radiolabeled ATP
08:05

Assaying Protein Kinase Activity with Radiolabeled ATP

Published on: May 26, 2017

Phosphorylation of the stress-activated protein kinase, MEKK3, at serine 166

Deanna G Adams1, Nancy A Sachs, Richard R Vaillancourt

  • 1Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721-0207, USA.

Archives of Biochemistry and Biophysics
|October 24, 2002
PubMed

Insights

MEKK3 protein phosphorylation was investigated, revealing Ser166 and Ser337 sites. These sites are regulated by various stimuli but do not affect MEKK3 activity or 14-3-3 protein binding.

Area of Science:

  • Cellular signaling pathways
  • Protein kinase regulation
  • Signal transduction mechanisms

Background:

  • Mitogen-activated protein kinase (MAPK) cascades are crucial in cellular responses.
  • The MEKK (MAP3K) family of protein kinases activates downstream MAPK pathways.
  • Direct phosphorylation and regulatory mechanisms of MEKK proteins remain largely uncharacterized.

Purpose of the Study:

  • To investigate the direct phosphorylation and regulation of MEKK3, a member of the MEKK family.
  • To identify upstream signaling pathways converging on MEKK3.
  • To determine the functional significance of MEKK3 phosphorylation.

Main Methods:

  • Recombinant (His)6FLAG.MEKK3 was expressed and purified from Sf9 insect cells.
  • Immobilized MEKK3 was used to precipitate interacting proteins, identified by liquid chromatography-mass spectrometry (LC-MS).
  • Phosphorylation sites were identified using LC-MS, and specific antibodies were generated to detect phosphorylated Ser166 and Ser337.

Main Results:

  • 14-3-3 proteins were identified as binding partners of MEKK3, suggesting serine phosphorylation.
  • Two specific phosphorylation sites, Ser166 and Ser337, were identified on MEKK3.
  • Stimuli including TNF, arsenite, forskolin, and serum induced phosphorylation of Ser166 and Ser337.
  • Neither Ser166 nor Ser337 phosphorylation was essential for 14-3-3 binding or MEKK3-mediated ERK/JNK activity.

Conclusions:

  • MEKK3 is phosphorylated at Ser166 and Ser337 in response to diverse extracellular stimuli.
  • These phosphorylation events do not appear to directly regulate MEKK3's interaction with 14-3-3 proteins or its kinase activity towards ERK and JNK.
  • MEKK3 functions as a convergence point for multiple upstream signaling pathways, integrating various cellular signals.

More Related Videos

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting10:08

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting

Published on: December 9, 2022

Oligopeptide Competition Assay for Phosphorylation Site Determination09:16

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

Related Concept Videos

MAPK Signaling Cascades01:07

MAPK Signaling Cascades

Mitogen-activated protein kinase, or MAPK pathway, activates three sequential kinases to regulate cellular responses such as proliferation, differentiation, survival, and apoptosis. The canonical MAPK pathway starts with a mitogen or growth factor binding to an RTK. The activated RTKs stimulate Ras, which recruits Raf or MAP3 Kinase (MAPKKK), the first kinase of the MAPK signaling cascade. Raf further phosphorylates and activates MEK or MAP2 Kinases (MAPKK), which in turn phosphorylates MAP...
PI3K/mTOR/AKT Signaling Pathway01:22

PI3K/mTOR/AKT Signaling Pathway

The mammalian target of rapamycin  (mTOR) is a serine/threonine kinase that regulates growth, proliferation, and cell survival in response to hormones, growth factors, or nutrient availability. This kinase exists in two structurally and functionally distinct forms: mTOR complex 1  (mTORC1) and mTOR complex 2  (mTORC2). The first form (mTORC1) is composed of a rapamycin-sensitive Raptor and proline-rich Akt substrate, PRAS40. In contrast,  mTORC2 consists of a rapamycin-insensitive companion...
Phosphorylation01:02

Phosphorylation

The addition or removal of phosphate groups from proteins is the most common chemical modification that regulates cellular processes. These modifications can affect the structure, activity, stability, and localization of proteins within cells as well as their interactions with other proteins.
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
Protein Kinases and Phosphatases02:54

Protein Kinases and Phosphatases

Proteins undergo chemical modifications that trigger changes in the charge, structure, and conformation of the proteins. Phosphorylation, acetylation, glycosylation, nitrosylation, ubiquitination, lipidation, methylation, and proteolysis are various protein modifications that regulate protein activity. Such modifications are usually enzyme-driven.
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
The JAK-STAT Signaling Pathway01:20

The JAK-STAT Signaling Pathway

Several cytokine receptors have tightly bound Janus kinase or JAK proteins attached at their cytosolic tail. Small signaling molecules such as cytokines, growth hormones, or prolactins bind to the cytokine receptors and initiate their dimerization. The dimerization brings the cytosolic JAKs together that trans-phosphorylate and activates each other. The activated JAKs now phosphorylate cytosolic tails of the cytokine receptors, which serve as binding sites for adaptor proteins such as  SH2...
cAMP-dependent Protein Kinase Pathways01:25

cAMP-dependent Protein Kinase Pathways

Cyclic Adenosine Monophosphate (cAMP) is an essential second messenger that activates protein kinase A (PKA) and regulates various biological processes. A single epinephrine molecule binds to GPCR and activates several heterotrimeric G proteins, each stimulating multiple adenylyl cyclase, amplifying the signal, and synthesizing large numbers of cAMP molecules. Small changes in cAMP concentration affect PKA activity. The binding of four cAMP molecules induces a conformational change in PKA,...